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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-24 to 2011-10-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD No. 471 and EU method B.13/14 in a GLP certified testing facility.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name: 400160
Batch no.: KL-11-038
Appearance: yellow, clear viscous liquid
Composition: UVCB, 2-Ethylhexyl esters of dimerised fatty acids, C18-unsaturated
Purity: 100% (GPC)
Production date: 24 March 2011
Expiry date: 24 March 2016
Storage: room temperature (20 ± 5 °C)
dissolved in ethanol

Method

Target gene:
Tester strains TA 98 and TA 97a are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Strain TA 102 is sensitive to oxidizing DNA damage based on AT base pairs at the site of the mutation.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA 97a
Additional strain / cell type characteristics:
other: Mutations in hisD6610, uvrB, pKM 101, and rfa
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: Mutations in hisD3052, uvrB, pKM 101, and rfa
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: Mutations in hisG46, uvrB, pKM 101, and rfa
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: Mutations in hisG428, pKM 101, and rfa
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: Mutations in hisG46, uvrB, and rfa
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate (S9 mix)
Test concentrations with justification for top dose:
plate incorporation method: 5002 / 1501 / 500 / 150 / 50 µg/plate
pre-incubation method: 5003 / 2502 / 1251 / 626 / 313 µg/plate
Vehicle / solvent:
Ethanol
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, H2O, ethanol
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylene diamine
Remarks:
concentration: 20 µg/plate; strains: TA 97a, TA 98 and TA 102 in the absence of metabolic activation
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
concentration: 1 µg/plate; strains: TA 100 and TA 1535 in the absence of metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-Amino-anthracene
Remarks:
concentration: 1 µg/plate; strain: TA 97a, TA 100, TA 102, TA 1535; with metabolic activation
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
concentration: 20 µg/plate; strain: TA 98; with metabolic activation
Details on test system and experimental conditions:
Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system.

Plate incorporation method:
In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium.

Pre-incubation method
In the pre-incubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium.
For both techniques, after 2 or 3 days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.
Evaluation criteria:
The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants minus mean spontaneous revertants) were calculated, too.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Statistics:
The use of statistics was limited to the calculation of means and standard deviations.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration related increase over the range tested can also be taken as a sign of mutagenic activity.
Remarks on result:
other: other: plate incorporation method and pre-incubation method
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1:    Mean No. of Revertants – Plate incorporation method

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

H2O

Mean

113

114

10

10

83

103

194

176

12

9

SD

11

7

2

2

14

9

29

3

1

2

DMSO

Mean

123

113

12

10

88

86

187

160

9

11

SD

10

14

2

2

10

14

22

7

2

3

Ethanol

Mean

120

113

12

13

110

117

180

228

15

17

SD

4

4

2

3

4

4

25

17

2

1

Pos.Contr.

Mean

476

510

242

231

510

472

857

795

227

136

SD

10

32

9

14

31

28

53

108

16

20

f(I)

3.87

4.51

20.17

23.10

6.14

5.49

4.58

4.97

18.92

12.36

5002 µg/pl.

Mean

101

107

10

11

99

93

197

200

11

11

SD

10

5

2

1

6

15

31

14

3

1

f(I)

0.84

0.95

0.83

0.85

0.90

0.79

1.09

0.88

0.73

0.65

1501 µg/pl.

Mean

109

102

11

13

104

86

182

205

10

13

SD

3

11

3

3

5

11

21

20

3

3

f(I)

0.91

0.90

0.92

1.00

0.95

0.74

1.01

0.90

0.67

0.76

500 µg/pl.

Mean

101

99

11

13

95

101

214

198

12

12

SD

8

6

2

2

6

12

11

12

2

2

f(I)

0.84

0.88

0.92

1.00

0.86

0.86

1.19

0.87

0.80

0.71

150 µg/pl.

Mean

106

117

12

11

99

90

188

194

13

13

SD

5

7

3

3

5

15

7

6

4

2

f(I)

0.88

1.04

1.00

0.85

0.90

0.77

1.04

0.85

0.87

0.76

50 µg/pl.

Mean

108

111

10

14

105

100

203

191

12

12

SD

6

6

2

4

2

10

9

8

2

2

f(I)

0.90

0.98

0.83

1.08

0.95

0.85

1.13

0.84

0.80

0.71

SD = standard deviation
f(I) = increase factor

Table 2     Mean No. of Revertants – Pre-incubation method

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

H2O

Mean

111

118

14

15

91

104

204

196

15

14

SD

9

7

3

4

9

10

18

12

1

1

DMSO

Mean

117

122

13

16

108

100

208

200

16

10

SD

5

6

4

3

14

9

22

21

1

3

Ethanol

Mean

111

111

15

17

102

112

218

195

12

14

SD

8

3

2

4

11

6

7

20

2

1

Pos.Contr.

Mean

723

677

377

305

530

547

510

519

282

315

SD

29

92

40

66

56

96

45

87

24

30

f(I)

6.18

5.55

29.00

19.06

5.82

5.47

2.45

2.60

18.80

31.50

5003 µg/pl.

Mean

117

120

12

12

85

102

202

211

15

14

SD

7

9

2

5

17

13

26

21

1

3

f(I)

1.05

1.08

0.80

0.71

0.83

0.91

0.93

1.08

1.25

1.00

2502 µg/pl.

Mean

109

114

12

12

78

79

210

202

14

16

SD

3

6

3

1

7

10

19

25

3

1

f(I)

0.98

1.03

0.80

0.71

0.76

0.71

0.96

1.04

1.17

1.14

1251 µg/pl.

Mean

117

117

11

10

84

91

188

235

17

16

SD

8

9

2

1

21

17

53

21

1

1

f(I)

1.05

1.05

0.73

0.59

0.82

0.81

0.86

1.21

1.42

1.14

626 µg/pl.

Mean

114

111

11

10

98

100

215

231

17

17

SD

3

3

2

1

35

8

8

23

1

1

f(I)

1.03

1.00

0.73

0.59

0.96

0.89

0.99

1.18

1.42

1.21

313 µg/pl.

Mean

121

120

11

10

82

81

206

196

16

17

SD

4

14

2

4

5

16

18

20

1

2

f(I)

1.09

1.08

0.73

0.59

0.80

0.72

0.94

1.01

1.33

1.21

SD = standard deviation

f(I) = increase factor

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

It can be stated, that under the test conditions, 400160 is not mutagenic in the bacterial reverse mutation test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102, and TA 1535.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 97, TA 98a, TA 100, TA 102, TA 1535 of Salmonella typhimurium were exposed to 400160 in sterile ethanol at concentrations of 5002 / 1501 / 500 / 150 / 50 µg/plate in the plate incorporation method and at concentrations of 5003 / 2502 / 1251 / 626 / 313 µg/plate in the pre-incubation method. Both test method were conducted in the presence and absence of mammalian metabolic activation.

400160 was tested at limit concentration (5000 µg/plate) for cytotoxicity. No cytotoxicity could be observed. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study was performed according to test guidelines OECD 471 and EU B.13/14 for bacterial reverse gene mutation data in a GLP certified testing facility.